1. Field of the Invention
The present invention relates generally to compositions for use in the preparation of Treponema pallidum antigenic proteins and peptides. In particular aspects, the invention relates to recombinant DNA constructs for the preparation of selected T. pallidum antigenic/immunogenic proteins and peptides.
2. Description of the Related Art
Treponema pallidum and its various pathogenic subspecies comprise the etiologic agents of syphilis and other treponemal diseases. Despite the availability of effective antibiotics, syphilis continues to result in worldwide morbidity and mortality. One of the principal reasons behind the continued medical threat posed by syphilis is the fact that this disease can go undiagnosed, and can continue to be transmitted sexually. Moreover, by going undetected or undiagnosed, the disease can exert extensive damage, which may not be resolved by therapy. This can be a particular problem in the case of cardiovascular syphilis, neurosyphilis, and even congenital forms of the disease.
Both prevention and diagnosis are important aspects of an overall approach to treponemal disease control. Particularly in places where some treponemal diseases tend to be endemic, such as Central and South America, Asia, or Africa, preventative measures such as immunization against the disease would likely provide the most reasonable and successful long term approach. Immunization of individuals against the disease through the use of vaccines incorporating particular T. pallidum pathogen-specific immunogenic proteins(s) is an approach which could be attempted. Unfortunately, no such vaccine currently exists.
The physician, in an attempt to make an accurate diagnosis with no definitive historical, epidemiological, or clinical evidence of the disease, relies totally upon serological results for accurate diagnosis. Unfortunately, the severe limitations and restrictions of the available serological procedures often preclude reliable diagnostic conclusions (83). It is well known that the immunologically non-specific non-treponemal screening tests for syphilis (VDRL, RPR circle card, ART) give false positive reactions not only among patients with acute and chronic diseases, but among pregnant women, vaccinees, and narcotic addicts (83-86). Furthermore, false negative reactions occur commonly among patients with untreated latent and late syphilis (83,87). Reliance, then, is placed upon the so-called "immunologically specific" confirmatory (treponemal) tests for syphilis (FTA-ABS, MEA-TP) to establish the presence or absence of the disease. Yet, these procedures also produce false positive reactions due to their technical and biological limitations, thereby creating the current diagnostic dilemma (83).
The lack of specificity of these tests stems from a two-fold problem. First, the preparation of either whole, killed T. pallidum (FTA-ABS test) or ultrasonic lysates of the organisms (MHA-TP test) requires organisms initially cultured in rabbit testes and thus necessitates the use of rabbit testis-contaminated T. pallidum. This contributes to false positive reactivity. Second, the presence of circulating cross-reactive antibody found in the serum of all humans (both patients and normal individuals) and elicited in response to the common antigens of host indigenous, non-pathogenic treponemes comprising the "normal bacterial flora" further complicates diagnostic accuracy. Although both the FTA-ABS and MHA-TP tests attempt to selectively bind these non-specific antibodies by the use of "sorbing" components prepared from one of the non-pathogenic treponemes (T. phagedenis biotype Reiter), their inability to effect a complete absorption of cross-reactive antibody results in false positive reactivities (83,88-92). Additionally, the use of "sorbing" reagents contributes to the complexity of the procedures.
Within the past decade, groups of investigators have begun to characterize on a molecular level the structural components of the Treponema pallidum, subspecies pallidum (T. pallidum), bacterium in an attempt to provide useful tools for both diagnosis and the prevention of syphilis (1). Certain of these investigators have sought to identify and to analyze "surface-associated" or "outer membrane" proteins of the organism in an attempt to define important immunogens or potential virulence determinants (1,2). For example, monoclonal antibodies directed against a 47-kilodalton (kDa) antigen of T. pallidum were reported to possess treponemicidal activity in the T. pallidum immobilization test and in the in vitro-in vivo neutralization test of Bishop and Miller (3,4). Moreover, further work demonstrated that this antigen was abundant in T. pallidum and highly immunogenic in both human and experimental rabbit syphilis (4-6). Even further evidence indicated that the 47-kDa antigen had potential as a serodiagnostic antigen and that monoclonal antibodies directed against the 47-kDa antigen may be used for syphilis diagnosis (5,7-13).
The 47-kDa antigen is not localized to just one pathogenic subspecies in that other pathogenic subspecies of treponemas such as T. pallidum subsp. pertenue, endemicum, and Treponema carateum all apparently possess cognate 47-kDa antigens (4,10,14-17); for example, the predominant serologic response in patients with active pinta (T. carateum) was found to be directed against the 47-kDa antigen (18). Immunologic, physicochemical, and genetic data support the pathogen-specificity of the 47-kDa antigen (4,10-12,17,19,20). Additional work on the 47-kDa antigen of T. pallidum by investigators other than the present inventor has recently been reviewed (11).
Up until the advent of the present invention, though, there has been no economical source for 47-kDa antigen. This is, of course, due to the requirement that T. pallidum can be grown only in connection with a living tissue, such as rabbit testicles. Moreover, in particular, there has been no recombinant DNA sources from which to prepare 47-kDa antigen proteins, peptides, or even specific DNA segments. Accordingly, an economic source for the provision of 47-kDa antigenic or immunogenic protein or peptides, a source free from possible contamination with animal or human viruses, would provide an important tool to be used in the diagnosis and/or possibly in the prevention of the disease.